Methods and compositions for treating and preventing arthritis

ABSTRACT

Methods and formulations are provided for treating or preventing arthritis, in particular osteoarthritis, where the formulation to be administered to a subject for treating or preventing arthritis comprises carvacrol, curcumin, epigallocatechin-3-gallate and oligomeric procyanidins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/520,156, filed Apr. 19, 2017, which is a U.S. national stage entryunder 35 U.S.C. § 371 of PCT International Patent Application No.PCT/US2015/058544, filed Nov. 2, 2015, which claims the benefit of U.S.Provisional Patent Application No. 62/076,842, filed Nov. 7, 2014, thecontents of each of which are incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberAR050968 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparentheses. Full citations for these references may be found at the endof the specification before the claims. The disclosures of thesepublications are hereby incorporated by reference in their entiretiesinto the subject application to more fully describe the art to which thesubject application pertains.

Arthritis is a leading cause of pain and disability. Osteoarthritis (OA)affects over 27 million Americans (1,2) and is a great economic burdenin the United States with over $185.5 billion in annual medical careexpenditures (3-7). The number of subjects suffering from arthritis isexpected to grow to over 67 million by 2030. The growth is credited toan increased aging population and increased prevalence of obesity.Within this population, 29.8% of the 45-64 age group reportdoctor-diagnosed arthritis and 50% of persons aged 65 or older arediagnosed with osteoarthritis. While 16.4% of under/normal weight adultsreport doctor-diagnosed arthritis, 52.5% of arthritis patients areoverweight or obese. Furthermore, patients who suffered from jointinjury currently account for 12% of all OA cases, which is approximately5.6 million in the United States.

Several treatments are available for osteoarthritis, including physicaltherapy, pharmacological therapies such as glucosamine sulfate,chondroitin sulfate, non-steroidal anti-inflammatory drugs (NSAIDs),corticosteroids, weak opioids or other analgesics, and finally end-stagedisease management and surgery (8). However, there is currently no curefor osteoarthritis nor do current therapies effectively slow or arrestits progression (5,9). Moreover, many of the pharmacological agents usedto treat osteoarthritis are associated with detrimentalgastrointestinal, renal, and cardiovascular side effects with long termuse (10-12). There are also many nutraceutical-based compounds (e.g.turmeric/curcumin, green tea, glucosamine, hyaluronic acid) marketed totreat joint pain and arthritis, but their effectiveness in treating OAhave not been clearly demonstrated, and none of these supplements havebeen approved by the FDA to treat or cure disease (13). While currenttherapeutic approaches may relieve pain and some disease symptoms, theymay fail to prevent or slow the progression of osteoarthritis becausethey do not adequately correct the pathological alterations in specificcellular and molecular pathways that are responsible for the disease.

The present invention addresses the need for improved treatments forarthritis, such as osteoarthritis and rheumatoid arthritis, where thetreatments are safe, effective and suitable for decades-long treatment.

SUMMARY OF THE INVENTION

The invention provides methods of treating or preventing arthritis, suchas osteoarthritis, in a subject in need thereof comprising administeringto the subject a formulation or composition comprising carvacrol,curcumin, epigallocatechin-3-gallate and oligomeric procyanidins in anamount effective to treat or prevent arthritis.

The invention also provides formulations or compositions for treatmentor prevention of arthritis comprising carvacrol, curcumin,epigallocatechin-3-gallate and oligomeric procyanidins, and apharmaceutically acceptable carrier.

This invention is superior to existing technologies because it arreststhe development of osteoarthritis, which no product currently on themarket can claim.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The C′-CEO formulation (combination of carvacrol, curcumin,epigallocatechin-3-gallate and oligomeric procyanidins) exerted highestefficacy in increasing expression of CITED2, a novelcartilage/chondrocyte protection molecular target, in comparison to theindividual components of C′-CEO. Human chondrocytes (C28/I2) treatedwith Carvacrol (C′, 1 μM), Curcumin (C, 1 μM), EGCG (E, 100 μM), OPC (0,50 μg/ml), or C′-CEO for 3 hours in the presence of IL-1β (10 ng/μl)were analyzed by real-time PCR. Bars represent mean CITED2expression±SEM. *p<0.05 using one-way ANOVA with Tukey post-hoc testcompared to control or indicated comparison), n=6/group.

FIG. 2A-2E. C′-CEO exerts the highest efficacy in suppressing expressionof cartilage degradation enzymes MMP-1 (A), MMP-3 (B), MMP-13 (C),ADAMTS5 (D) and pro-inflammatory mediator TNF-α (E), in comparison tothe individual components of C′-CEO. Human chondrocytes (C28/I2) treatedwith Carvacrol (C′, 1 μM), Curcumin (C, 1 μM), EGCG (E, 100 μM), OPC (O,50 μg/ml), or C′-CEO (combination of all compounds) for 3 hours in thepresence of IL-10 (10 ng/μl), a cell culture model of osteoarthritis.Data were analyzed by real-time PCR. Bars represent mean gene expressionof MMP-1, -3, -13, ADAMTS5, TNF-α±SEM. *p<0.05 using one-way ANOVA withTukey post-hoc test compared to control or indicated comparison,n=6/group.

FIG. 3. Mice treated with C′-CEO formulation exhibited the least amountof histologic cartilage damage compared to individual components ofC′-CEO. Carvacrol (C′: 50 mg/kg), Curcumin (C: 50 mg/kg), EGCG (E: 5mg/kg), OPC (O: 50 mg/kg), C′-CEO (combination of all compounds) orvehicle were administered daily (7 days/week) for 8 weeks via oralgavage in DMM mice (C57BL/6, male, 6 mo., n=8/group), with naïve as anadditional control. OA severity based on Safranin 0 staining.

FIG. 4A-4B. The C′-CEO formulation prevents breakdown of the cartilageextracellular matrix, suppresses proteolytic enzymes MMP-13 and ADAMTS5,and increases expression of CITED2 in an OA mouse model. A.Immunohistochemistry for denatured type II collagen (Col2 3/4M), cleavedaggrecan (NITEGE), MMP-13 and ADAMTS-5 in the articular cartilage ofvehicle or C′-CEO-treated DMM mice and naïve controls. B.Immunohistochemistry for CITED2 and % of CITED2 positive chondrocytes inthe articular cartilage of vehicle or C′-CEO-treated DMM mice and naïvecontrols.

FIG. 5A-5C. C′-CEO exerted the greatest effect on pain relief usingwell-established pain assessments in a post-traumatic OA animal model.Arthritic pain was assessed in DMM OA mice treated as described in FIG.3. At 8 weeks after DMM, OA-associated pain was assessed using assays of(A) von Frey (mechanical allodynia), and (B) distance traveled and (C)hind limb rearing. The von Frey test consisted of exposing the hind pawto von Frey filaments in ascending order. Groups were evaluated usingnumber of withdrawal responses normalized to their baseline. The openfield behavioral test measured the distance (cm) traveled and timesreared (standing on hind limbs) in 6 min. Bars represent meanvalues±SEM. *p<0.05 using one-way ANOVA with Tukey post-hoc testcompared to control or indicated comparison, n=8/group.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of treating or preventing arthritis in asubject in need thereof comprising administering to the subject aformulation or composition comprising carvacrol, curcumin,epigallocatechin-3-gallate and oligomeric procyanidins in an amounteffective to treat or prevent arthritis.

The arthritis can be, for example, osteoarthritis, which can arise forexample subsequent to injury to a joint. In another embodiment, thearthritis can be, for example, rheumatoid arthritis.

As used herein, to “treat” arthritis means to ameliorate a sign and/orsymptom of arthritis and/or to arrest the development of arthritis.Preferably, administration of the formulation or composition to thesubject arrests or slows the breakdown of joint tissue affected byarthritis. Preferably, administration of the formulation or compositionto the subject reduces inflammation associated with arthritis.Preferably, administration of the formulation or composition to thesubject relieves painful symptoms of arthritis. Preferably,administration of the formulation or composition to the subject improvesone or more of sensitivity to mechanical stimuli, joint movement andjoint function.

To “prevent” arthritis means to prevent the development of arthritis ina subject at risk for development of arthritis. In one embodiment, forexample, the subject has sustained a joint injury and is taking theformulation or composition to prevent development of post-traumaticosteoarthritis.

The invention also provides a formulation or composition for treatmentor prevention of arthritis comprising four nutraceuticals: Carvacrol(C′), Curcumin (C), Epigallocatechin-3-gallate (E), and Oligomericprocyanidins (O) (“C′-CEO” formulation or composition) and apharmaceutically acceptable carrier. In one embodiment, the activeingredients of the formulation or composition consist of carvacrol,curcumin, epigallocatechin-3-gallate and oligomeric procyanidins.

As used herein, a “pharmaceutically acceptable carrier” is (i)compatible with the other ingredients of the composition withoutrendering the composition unsuitable for its intended purpose, and (ii)suitable for use with subjects as provided herein without undue adverseside effects (such as toxicity, irritation, and allergic response). Sideeffects are “undue” when their risk outweighs the benefit provided bythe composition. Non-limiting examples of pharmaceutically acceptablecarriers include any of the standard pharmaceutical carriers such asphosphate buffered saline solutions, water, and emulsions such asoil/water emulsions and microemulsions.

The formulation or composition can be administered to subjects usingroutes of administration known in the art. Preferred routes ofadministration include oral administration, intra-articular injection,and topical administration. One or more of the components of the C′-CEOformulation can be nano-encapsulated to improve its solubility. TheC′-CEO formulation can be encapsulated in enteric-coated capsules fororal delivery to limit gastrointestinal degradation.

In one embodiment, the molar ratio of carvacrol, curcumin, EGCG and OPCin the formulation or composition is 1:1:100:50. In one embodiment, theweight ratio of carvacrol, curcumin, EGCG and OPC in the formulation orcomposition is 10:10:1:10.

This invention will be better understood from the Experimental Detailsthat follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS Materials and Methods

Induction of osteoarthritis in mice.

All studies were approved by the Albert Einstein College of MedicineInstitutional Animal Care and Use Committee. Destabilization of themedial meniscus (DMM) was established in adult C57BL/6 mice (male, 5-6months) by surgically transecting the medial meniscotibial ligament(MMTL) in the right hind limb (36). Briefly, the joint capsuleimmediately medial to the patellar tendon was incised, followed by bluntdissection of the fat pad, to provide visualization of the MMTL of themedial meniscus. The MMTL was transected, leading to DMM. In the shamsurgery, the MMTL was visualized but not transected. The joint capsuleand skin were closed with suture.

Immunohistochemistry, Safranin O Staining, and OARSI Score Evaluation.

Animals were euthanized, hindlimbs were fixed in formalin, decalcifiedin formic acid, embedded in paraffin and sectioned for histology andimmunohistochemistry. Sections were incubated overnight at 4° C. withantibodies against cleaved aggrecan (NITEGE, Ibex) and cleaved type IIcollagen (Col2-3/4M, Ibex), MMP-13 (Abcam), and ADAMTS5 (Abcam) followedby incubation with anti-mouse or anti-rabbit secondary antibody (BiocareMedical) and visualization with DAB chromagen (Vector Laboratories).Negative controls were stained with irrelevant isotype-matchedantibodies (Biocare Medical). Safranin 0-fast green staining was used tovisualize proteoglycans in the articular cartilage. Severity of OA wasevaluated for each mouse using the Osteoarthritis Research SocietyInternational (OARSI) scoring system (34). Immunostaining intensity fortype II collagen or aggrecan cleavage epitopes was quantified bydetermining the “reciprocal intensity” of the stained articularcartilage matrix. Briefly, the light intensity value of random locationswithin azones from the posterior to anterior direction of the femoraland tibial condyles was measured using the color picker in AdobePhotoshop (37). Percentages of positive MMP-13 and ADAMTS5 chondrocyteswere determined by counting the number of immunostained cells anddividing by the total number of chondrocytes visualized by a hematoxylincounterstain (Vector Laboratories).

Tactile Sensitivity Testing.

Mice were acclimated for 30 minutes in individual chambers on top of awire grid platform prior to von Frey testing. The plantar surface of thehind paw was stimulated with ascending force intensities of von Freyfilaments (Stoelting) to determine tactile sensitivity. A positiveresponse was defined as a rapid withdrawal of the hind paw when thestimulus was applied, and the number of positive responses for eachstimulus was recorded. Tactile threshold was defined as a withdrawalresponse in 5 out of 10 trials to a given stimulus intensity (38). Thisthreshold was calculated once per animal.

Open Field Behavioral Test.

Mice were acclimated to the test room for 30 min before open fieldtesting. Mice were placed in the center of individual plexiglass squarechambers (45 cm×45 cm) and allowed to freely explore the chamber for theduration of the test session. The movements of the mice were recordedwith a video camera. Upon completion of the test, each mouse wasreturned to its home cage (39). Mouse movements were manually traced tocalculate the distance (in cm) the mouse traveled within the cage in 6min. The number of times each mouse reared (standing on its hind limbs)within 6 min was recorded by two observers blinded to treatment groupassignments.

Results

Selection of Components of C′-CEO Formulation.

The C′-CEO formulation was identified by an innovative screeningstrategy; it is formulated from nutraceutical compounds that induce theexpression of a chondroprotective transcriptional regulator CITED2(Cbp/p300 Interacting Transactivator with ED-rich tail 2), a potentialosteoarthritis therapeutic target (29-32). CITED2 mediates a novelpathway that plays a critical role in chondroprotection, at least inpart by suppressing expression and activity of cartilage degradingenzymes MMP-13 and ADAMTS-5 (29, 31). Notably, CITED2 deficiency isassociated with degradation of human osteoarthritic cartilage andpost-traumatic osteoarthritic mice. Experimental reduction of CITED2expression in synovial joint tissues causes osteoarthritis (29, 31).Gene transfer of CITED2 effectively slows post-traumatic diseaseinitiation and progression in mice (30).

Because of the chronic nature of osteoarthritis and the need forlong-term therapies, the inventors searched for activators of CITED2—anovel target for osteoarthritis treatment among non-toxic nutraceuticalcompounds. It was first found that green tea extract EGCG suppressesMMP-13 and ADAMTS-5 by inducing CITED2 (35). To enhance the potency ofthis potential drug, three other compounds were identified thatsynergistically enhance CITED2 induction and other anti-arthriticeffects. The C′-CEO formulation is a product designed to exhibit bothdisease-modifying and symptom-modifying properties and to be effectivefor both prevention and treatment of arthritis such as osteoarthritis.

Effect of the C′-CEO Formulation on Expression of CITED2, a NovelCartilage/Chondrocyte Protection Molecular Target.

The experiments were conducted in an in vitro OA model—chondrocytecultured in the presence of pro-inflammatory cytokine IL-1β, whichmimics OA conditions in humans. This system has been well recognized andis commonly used as validation of drug screening for OA. Humanchondrocytes (C28/I2) were treated with Carvacrol (C′, 1 μM), Curcumin(C, 1 μM), EGCG (E, 100 μM), OPC (O, 50 μg/ml), or C′-CEO (combinationof all compounds) for 3 hours in the presence of IL-10 (10 ng/μl), acell culture model of osteoarthritis. Data were analyzed by real-timePCR.

C′-CEO compared to each individual compound carvacrol (C′), curcumin(C), EGCG (E) and Oligomeric Proanthocyanidins (O) exerted the highestincrease in expression of CITED2 (FIG. 1). The C′-CEO formulationsynergistically increased expression of CITED2 to a degree higher thaneach individual compound alone.

Effect of C′-CEO Formulation on Expression of Proteolytic Enyzmes (MMPs1, 3, 13, ADAMTS5), and Pro-Inflammatory Mediator TNF-α, which PlayCritical Roles in OA Development.

To determine the potential therapeutic efficacy of C′-CEO, the abilityof the C′-CEO formulation to reduce expression of proteolytic enzymesand a pro-inflammatory cytokine was tested in vitro. Proteolytic enzymessuch as matrix metalloproteinases (MMP)-1, -3, -13, and A DisintegrinAnd Metalloproteinase with Thrombospondin Motifs (ADAMTS5) are highlyexpressed in arthritic cartilage, and directly cleave the cartilageextracellular matrix. Mice with MMPs or ADAMTS knocked out have beendemonstrated to be resistant to the development of OA, suggestingtargeting these enzymes will have therapeutic benefit. In addition,expression of pro-inflammatory cytokines, such as IL-1β and TNF-α areincreased in arthritic tissue, and upregulate expression of MMPs/ADAMTS.

Human chondrocytes (C28/I2) were treated with Carvacrol (C′, 1 μM),Curcumin (C, 1 μM), EGCG (E, 100 μM), OPC (O, 50 μg/ml), or C′-CEO(combination of all compounds) for 3 hours in the presence of IL-1β (10ng/μl), a cell culture model of osteoarthritis. Data were analyzed byreal-time PCR.

Each individual compound exerted some efficacy in suppressing expressionof MMP-1, 3, 13, ADAMTS5, and pro-inflammatory cytokine TNF-α. However,overall, C′-CEO was most the effective in suppressing expression ofthese targets, compared to each individual compound and vehicle control(FIG. 2). C′-CEO effectively suppressed expression of multiple cartilagedegradative enzymes and a pro-inflammatory mediator, suggesting it hastherapeutic potential for treating OA.

Efficacy of C′-CEO Formulation In Vivo in Slowing Progression of OA.

To determine the efficacy of the C′-CEO formulation in slowingprogression of OA in vivo, a well-established post-traumatic mouse modelof OA was used. These OA mice were orally administered C′-CEO,individual compounds, or placebo daily. Severity of OA was evaluated 8weeks after induction of OA, using a well-established and FDArecommended cartilage integrity stain, Safranin O, and asemi-quantitative Osteoarthritis Research Society International (OARSI)scoring system. Furthermore, the efficacy of the C′-CEO formulation wasvalidated by examining the level of breakdown of two major cartilagematrix proteins, type II collagen and aggrecan, as cartilage degradationis a hallmark of OA. It was further examined how the C′-CEO formulationmay have such effect by targeting CITED2 by examining CITED2 expressionin the articular cartilage of these OA mice.

Since as described above, the C′-CEO formulation was more effective thanits individual components in increasing expression of CITED2 and insuppressing expression of major proteolytic enzymes and apro-inflammatory mediator involved in OA initiation and development, theefficacy of the C′-CEO formulation was determined in slowing progressionof OA in a post-traumatic OA mouse model.

All studies were approved by the Albert Einstein College of MedicineInstitutional Animal Care and Use Committee. Destabilization of themedial meniscus (DMM) was established in adult C57BL/6 mice (male, 5-6months) by surgically transecting the medial meniscotibial ligament(MMTL) in the right hind limb. Immediately after surgery, mice wereadministered either carvacrol (C′), curcumin (C), EGCG (E), OPC (O),C′-CEO, or vehicle control for 8 weeks. At 8 weeks following surgery,animals were euthanized, the hindlimbs were fixed in formalin,decalcified in formic acid, embedded in paraffin and sectioned forhistology. Safranin O, a well-established cartilage stain, was used tovisualize integrity of the articular cartilage and aggrecans in thearticular cartilage. Severity of OA was evaluated using the OARSIscoring system.

While the individual compounds—carvacrol (C′), curcumin (C), EGCG (E),and OPC (O), exhibited some reduction in cartilage degradation, mice fedthe C′-CEO formulation exhibited the least amount of cartilage damage(fibrillation, erosion, loss of aggrecans), as determined histologicallywith Safranin O staining (FIG. 3). The cartilage of C′-CEO mice alsoexhibited quantitatively the lowest OA score, established by an OAscoring system (OARSI score), compared to treatment with each individualcompound alone (Table 1). Thus, while treatment with the individualcompounds exerted some efficacy in cartilage protection, treatment withthe combination of all four nutraceuticals, the C′-CEO formulation,exerted the greatest efficacy in slowing progression of OA in apost-traumatic OA animal model.

Efficacy of C′-CEO Formulation on Preventing the Breakdown of theCartilage Extracellular Matrix, a Hallmark of OA, and in IncreasingCITED2 Expression In Vivo.

The cartilage extracellular matrix consists primarily of two proteins:type II collagen and aggrecan. Breakdown of these matrix proteins is ahallmark of OA. Therefore, the efficacy of the C′-CEO formulation onpreventing breakdown of these proteins, and also its effect on theexpression of CITED2, the molecular target of C′-CEO, were examined.

The articular cartilage of C′-CEO-treated mice exhibited less positiveimmunostaining for cleaved type II collagen (Col2 3/4M) and cleavedaggrecan (NITEGE), when compared to vehicle-treated mice. The number ofchondrocytes expressing MMP-13 and ADAMTS5, which cleave type IIcollagen and aggrecan, respectively, was also examined. C′-CEO-treatedmice had fewer chondrocytes expressing MMP-13 and ADAMTS in thearticular cartilage, when compared to vehicle controls (FIG. 4A). Tissuesections from the hind limbs of experimental animals were incubatedovernight at 4° C. with antibodies against cleaved aggrecan (NITEGE,Ibex) and type II collagen (Col2-3/4M, Ibex), MMP-13 (Abcam), andADAMTS5 (Abcam) followed by incubation with anti-mouse or anti-rabbitsecondary antibody (Biocare Medical) and visualization with DABchromagen (Vector Laboratories). Negative controls were stained withirrelevant isotype-matched antibodies. C′-CEO-treated mice alsocontained a higher percentage of chondrocytes expressing CITED2 (FIG.4B). The C′-CEO formulation may exert efficacy in cartilage protectionby slowing degradation of the two main cartilage extracellular matrixproteins (type II collagen and aggrecan), and upregulating CITED2 in thearticular cartilage.

Efficacy of the C′-CEO Formulation in Relieving OA-Related Pain in aWell-Established Post-Traumatic OA Animal Model.

The progression of OA is accompanied by secondary clinical symptoms,most prominently pain. In addition to evaluating the disease-modifyingeffect of the C′-CEO formulation, it was determined whether C′-CEOexerted pain relief in this OA animal model, at 8 weeks following OAinduction. To determine pain relief, three animal behavior assessmentswere used: 1) von Frey, which determined sensitivity to mechanicalstimuli [OA animals tend to have lower thresholds of pain to mechanicalstimuli]; 2) distance traveled—a measure of joint movement [OA animalstend to move a lesser distance compared to non-OA animals]; 3) rearing—ameasure of joint function [OA animals tend to rear, or stand up on theirhind limbs less than non-OA animals].

Treatment with the individual compounds, compared to the vehicle controldid not improve sensitivity of the mice to mechanical stimuli (FIG. 5A),did not increase distance traveled (FIG. 5B), and did not increase hindlimb rearing (FIG. 5C). In contrast, C′-CEO-treated DMM mice, like naïvecontrols, required a higher force to elicit paw withdrawal (p<0.05, FIG.5A), traveled a longer distance (p<0.05, FIG. 5B), and increasedrearing/standing on their hind limbs (p<0.05, FIG. 5C).

Treatment through oral administration with the individual compounds ofcarvacrol, curcumin, EGCG, or OPC did not result in a statisticalimprovement in pain relief of OA-associated pain. However,C′-CEO-treated OA mice exhibited statistical improvements in painrelief, with levels of mechanical sensitivity, joint movement andfunction, on levels similar to naïve, non-OA animals.

TABLE 1 Mice treated with C'-CEO formulation exhibited the lowest OAhistologic score. OARSI score is a semi- quantitative histologic-basedscoring system with 0 indicating healthy cartilage and 6 representingsevere cartilage erosion. The mean OARSI score of 8 animals/group ispresented ± standard deviation. *p < 0.05, one-way ANOVA compared tovehicle-treated animals. ** p < 10.05, one-way ANOVA compared toindividual-treatments. C'-CEO vs. Carvacrol (C') Vehicle 4 ± 0.5 Carvacrol 2.9 ± 0.45* C'-CEO 1.75 ± 0.24** C'-CEO vs. Curcumin (C)Vehicle 4 ± 0.5  Curcumin 2.4 ± 0.42* C'-CEO 1.75 ± 0.24** C'-CEO vs.EGCG (E) Vehicle 4 ± 0.5  EGCG 2.1 ± 0.29* C'-CEO 1.75 ± 0.24** C'-CEOvs. OPC (O) Vehicle 4 ± 0.5  OPC 2.3 ± 0.32* C'-CEO 1.75 ± 0.24**

DISCUSSION

The present results provide clear evidence that in vivo the inventedC′-CEO formulation exerts statistically significant synergistic effectsnot only on osteoarthritis disease initiation and progression, but alsoon relieving pain symptoms and improving mobility, when compared to eachindividual treatment alone and to vehicle controls. The C′-CEOformulation significantly slowed development of post-traumaticosteoarthritis when oral administration was begun immediately after DMMinjury in mice. Preventing or reducing the severity of osteoarthritis isextremely valuable because tissue damage is very difficult to reverse.About 50% of patients who suffer ACL injuries develop osteoarthritiswithin 10-15 years (15). An oral preventative would be convenient forpatients who have suffered an ACL tear or meniscal injuries and areotherwise asymptomatic.

The C′-CEO formulation may not only modify the course of the diseasedirectly, but also relieve pain symptoms and improve mobility. Resultsin studies with post-traumatic osteoarthritis mice makes the C′-CEOformulation potentially extremely valuable because pain relief andimproved mobility are significant goals of clinical treatments.Uncontrolled pain is a primary reason why patients chosejoint-replacement surgery (16).

The C′-CEO formulation may also be used to treat other forms ofarthritis including rheumatoid arthritis. Inflammation, cartilagedegradation and chronic pain are common hallmarks of all forms ofarthritis including osteoarthritis and rheumatoid arthritis (17). Theanti-inflammatory, anti-catabolic and analgesic actions of the C′-CEOformulation are likely to have beneficial effects on all forms ofarthritic disease.

The components of the C′-CEO formulation were selected by molecularscreening for the ability to stimulate production of a regulatoryprotein having a range of cartilage-maintaining (chondroprotective)properties. These include suppressing the expression of severaltissue-degrading enzymes implicated in the pathophysiology ofosteoarthritis. The components of the C′-CEO formulation are individualnutraceutical compounds that induce a chondroprotective transcriptionalregulator CITED2 (Cbp/p300 Interacting Transactivator with ED-rich tail2), a potential OA therapeutic target (29-32). These studies suggestthat CITED2 not only modulates signal pathways related to chondrocytehomeostasis, but also protects cartilage integrity.

In addition, the components of the C′-CEO formulation were expresslychosen from a class of food products (“nutraceuticals”) that are likelyto be well tolerated over long periods of chronic treatment.Nutraceuticals, as food or diet supplements, are traditionallyconsidered to provide medical or health benefits and to be potentiallysafe alternatives to standard pharmacologic therapies (18-27). Thenutraceuticals proposed for osteoarthritis treatment are derived in somecases from traditional alternative medicine (28) or based on theirgeneral anti-inflammatory effects (18). Before the present study, theeffectiveness of nutraceuticals in preventing or slowing osteoarthritisprogression has not been well established (13).

The compounds that constitute the C′-CEO formulation are regarded assafe (GRAS) by the FDA. This is particularly important as osteoarthritishas become more prevalent in adults over 40 years and risk factorsincluding joint injuries, obesity and diabetes may lead to diseasedevelopment at even younger ages (14). Non-toxic nutraceuticals can beused safely in individual patients over the decades necessary formeaningful benefit (13).

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What is claimed is:
 1. A method of treating arthritis in a subject inneed thereof, comprising administering to the subject as activeingredients: a therapeutically-effective amount of carvacrol; atherapeutically-effective amount of curcumin; atherapeutically-effective amount of epigallocathein-3-gallate; and atherapeutically-effective amount of oligomeric procyanidins.
 2. Themethod of claim 1, wherein the active ingredients are in apharmaceutical formulation and the pharmaceutical formulation furthercomprises a pharmaceutically acceptable carrier.
 3. The method of claim1, wherein the carvacrol, the curcumin, the epigallocathein-3-gallate,and the oligomeric procyanidins are present in a molar ratio of1:1:100:50.
 4. The method of claim 1, wherein the carvacrol, thecurcumin, the epigallocathein-3-gallate, and the oligomeric procyanidinsare present in a weight molar of 10:10:1:10.
 5. The method of claim 1,wherein the active ingredients are synergistic.
 6. The method of claim1, wherein the administering is by injection.
 7. The method of claim 1,wherein the administering is topical.
 8. The method of claim 1, whereinthe administering is oral.
 9. The method of claim 1, wherein theadministering reduces cartilage degradation associated with thearthritis in the subject.
 10. The method of claim 1, wherein theadministering reduces inflammation associated with the arthritis in thesubject.
 11. The method of claim 1, wherein the administering reducespain symptoms associated with the arthritis in the subject.
 12. Themethod of claim 1, wherein the administering increases expression ofCbp/p300 interacting transactivator with ED-rich tail 2 (CITED2) in thesubject.
 13. The method of claim 1, wherein the administering suppressesexpression of a cartilage degradation enzyme in the subject.
 14. Themethod of claim 1, wherein the administering suppresses expression ofmatrix metalloproteinase-1 (MMP-1) in the subject.
 15. The method ofclaim 1, wherein the administering suppresses expression of matrixmetalloproteinase-3 (MMP-3) in the subject.
 16. The method of claim 1,wherein the administering suppresses expression of matrixmetalloproteinase-13 (MMP-13) in the subject.
 17. The method of claim 1,wherein the administering suppresses expression of ADAM metallopeptidasewith thrombospondin type 1 motif 5 (ADAMTS5) in the subject.
 18. Themethod of claim 1, wherein the administering suppresses expression oftumor necrosis factor-alpha (TNF-α) in the subject.
 19. The method ofclaim 1, wherein the arthritis is osteoarthritis.
 20. The method ofclaim 1, wherein the arthritis is rheumatoid arthritis.